The invention disclosed herein relates generally to inhibitors of novel, functional GPCR heteromers, and more specifically to inhibitors of CXC receptor 4 (CXCR4)-G protein-coupled receptor (GPCR) heteromers that display enhanced signaling downstream of CXCR4 as a result of functional heteromer formation and that are associated with cancers and other diseases.
G protein-coupled receptors (GPCRs) are seven-transmembrane domain cell surface receptors that are coupled to G proteins. GPCRs mediate diverse sensory and physiological responses by perceiving stimuli including light, odorants, hormones, neurotransmitters, chemokines, small lipid molecules, and nucleotides. There are approximately 800 GPCR genes in human genome, and more than half of them are predicted to encode sensory receptors such as olfactory, visual, and taste receptors (Bjarnadottir, et al., 2006). The remaining 350 GPCRs have physiologically important roles in embryonic development, behavior, mood, cognition, regulation of blood pressure, heart rate, and digestive processes, regulation of immune system and inflammation, maintenance of homeostasis, and growth and metastasis of cancers (Filmore 2004, Overington, et al., 2006). GPCRs are associated with many diseases and are the targets of approximately 40% of all prescription drugs (Filmore 2004).
CXC receptor 4 (CXCR4) is a member of the chemokine receptor family GPCR. CXCR4 is expressed on most of the hematopoietic cell types, bone marrow stem cells, endothelial progenitor cells, vascular endothelial cells, neurons and neuronal stem cells, microglia and astrocytes (Klein and Rubin 2004, Griffith, et al., 2014). CXCR4 responds to its ligand C-X-C Motif Chemokine ligand 12 (CXCL12), also known as Stromal cell-derived factor 1 (SDF-1), and has essential roles in the embryonic development of the hematopoietic, cardiovascular, and nervous systems (Griffith, et al., 2014). CXCR4 was discovered as a co-receptor for human immunodeficiency virus (HIV), and has important roles in the homing of hematopoietic stem cells (HSCs) to the bone marrow, inflammation, immune surveillance of tissues, and tissue regeneration in adult (Chatterjee, et al. 2014). Mutations in the C-terminus of CXCR4 cause persistent CXCR4 activation, leading to a congenital immune deficiency called WHIM syndrome (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) characterized by neutropenia and B cell lymphopenia (Hernandez, et al., 2003; Kawai, 2009). CXCR4 also has essential roles in T and B lymphocyte development within lymphoid organs and the thymus during development and in adult (Allen, et al., 2004; Ara, et al., 2003).
CXCR4 is implicated in various immune and autoimmune diseases, such as HIV infection, ischaemia, wound healing, rheumatoid arthritis, systemic lupus erythematosus (SLE), interstitial pneumonias, vascular disease, multiple sclerosis, pulmonary fibrosis, and allergic airway disease (Chu et al., 2017; Debnath, et al., 2013; Domanska, et al., 2013). The involvement of CXCR4 in rheumatoid arthritis was demonstrated by the increased accumulation of CXCR4-positive T-cells in arthritic joints, and a reduction in collagen-induced arthritis in CXCR4-deficient mice (Buckley et al., 2000; Chung et al., 2010). Moreover, CXCR4 antagonist, AMD3100, alleviated collagen-induced arthritis significantly in a mouse model (De Klerck et al., 2005). CXCR4 also regulates pulmonary fibrosis by recruiting circulatory fibroblasts and bone marrow-derived progenitor cells during lung injury, and AMD3100 demonstrated a preventive effect in bleomycin-induced mouse pulmonary fibrosis (Song et al., 2010). CXCR4/CXCL12 axis is also implicated in the pathogenesis of SLE. In mouse models of lupus and patients with SLE, inflammatory cells such as monocytes, neutrophils, and B-cells showed increased expression of CXCR4 and migrated toward skin and lung that overexpress CXCL12 predominantly (Chong and Mohan, 2009; Wang et al., 2009; Wang et al., 2010). CXCR4 antagonist, CTCE-9908, prolonged survival and greatly improved disease conditions and nephritis in a mouse model of lupus (Wang et al., 2009). Furthermore, CXCR4 is also involved in brain and cardiac diseases including brain injury, stroke, myocardial infarction, atherosclerosis and injury-induced vascular restenosis (Cheng et al., 2017; Domanska, et al., 2013; Doring, et al., 2014).
The involvement of CXCR4 in cancer was first noticed when B cells from patients with chronic lymphocytic leukemia (B-CCL) express high levels of functional CXCR4 on the surface, showing enhanced calcium mobilization and actin polymerization upon CXCL12 exposure, and migration towards bone marrow stromal cells that secret CXCL12 (Burger, et al., 1999).
CXCR4 is subsequently characterized to be responsible for breast cancer metastasis to organs that express higher levels of CXCL12 such as lymph nodes, bone marrow, lung, and liver (Muller, et al., 2001). After initial discovery, increasing evidence indicates that CXCR4 is associated with a variety of different cancers and has multiple potential roles in malignancy. CXCR4 is overexpressed in more than 23 human cancers, including breast cancer, lung cancer, brain cancer, kidney cancer (or renal cell carcinoma), pancreatic cancer, ovarian cancer, prostate cancer, melanoma, leukemia, multiple myeloma, gastrointestinal cancers, and soft tissue sarcomas, and regarded as a poor prognosis marker (Domanska, et al., 2013; Chatterjee, et al., 2014; Furusato et al., 2010). CXCR4 is the only chemokine receptor that is expressed by the majority of cancer types (Liang et al., 2015), and stimulates tumor cell growth, survival, and invasiveness in response to CXCL12 secreted by the cancer cells and the surrounding cancer-associated cells (Burger and Kipps, 2006; Chatterjee et al., 2014; Domanska et al., 2013).
Systematic meta-analyses using databases including PubMed, EMBASE, and Cochrane library indicate significant association between CXCR4 over-expression and poorer progression-free survival and overall survival in various cancers including hematological malignancy, breast cancer, colorectal cancer, esophageal cancer, head and neck cancer, renal cancer, lung cancer, gynecologic cancer, liver cancer, prostate cancer, and gallbladder cancer (Du et al., 2015; Hu et al., 2015; Li et al., 2017; Wang et al., 2016; Zhao et al., 2015).
CXCR4/CXCL12 axis plays a central role in tumor growth, invasion, angiogenesis, vasculogenesis, metastasis, drug resistance, and cancer cell-tumor microenvironment interaction (D'Alterio et al., 2012; Domanska et al., 2013; Guo et al., 2016)
The critical role of CXCR4 in cancer cell proliferation and tumor growth was demonstrated in various experimental models in vitro and in vivo such as orthotopic, subcutaneous human xenograft, and transgenic mouse models using CXCR4 antagonists (Domanska et al., 2013). The Daoy medulloblastoma cells and U87 glioblastoma cells showed CXCR4 expression and exhibited dose-dependent increase in proliferation to a gradient of CXCL12 in vitro, and systemic administration of AMD3100 inhibited the growth of intracranial U87 and Daoy cell xenografts (Rubin et al., 2003).
Involvement of CXCR4 in the metastasis of cancer cells towards CXCL12 expressing organs was also demonstrated in pancreatic, thyroid, melanoma, prostate, and colon cancer xenograft models (Bartolome et al., 2009; De Falco et al., 2007; Taichman et al., 2002; Wang et al., 2008; Zeelenberg et al., 2003).
The main mechanism of action described for the small molecules or peptides antagonists of CXCR4 is centered on their ability to mobilize malignant cells from the BM, thereby sensitizing them to chemotherapy. These agents have shown limitations regarding short half-lives, making their adequate management over long periods of time difficult (Hendrix et al., 2000). In contrast, therapeutic monoclonal antibodies have the advantage of having more prolonged half-lives, and are suitable for less frequent dosing. Additionally, human IgG antibodies have the ability to induce cell death upon binding to their target protein on cancer cells, via interaction with Fc-receptor on effector cells, including antibody-dependent cell mediated cytotoxicity/phagocytosis (ADCC/ADCP) (Jiang et al., 2011). Such cytotoxic mechanism of action are not inherent to small molecules or peptides, and have been demonstrated to play a key role in the clinical activity of several therapeutic antibodies (Wang et al., 2015).
Targeting CXCR4 using a neutralizing anti-CXCR4 antibody or CXCR4 specific antagonists inhibited primary tumor growth as well as metastasis to secondary organs in breast cancer, colon cancer, hepatocellular carcinoma, osteosarcoma, and melanoma (De Falco et al., 2007; Hassan et al., 2011; Huang et al., 2009; Kim et al., 2008; Muller et al., 2001; Schimanski et al., 2006; Smith et al., 2004; Zeelenberg et al., 2003). In a transgenic breast cancer mouse model, inhibition of CXCR4 with CTCE-9908 reduced not only the growth of primary tumor but also the expression of vascular endothelial growth factor (VEGF) and AKT phosphorylation (Hassan et al., 2011).
Cancer stem cells (CSCs) are a population of cancer cells with properties such as infinite self-renewal, potential to differentiate into multiple cancer lineages, ability to adopt a quiescent state, and intrinsic high resistance to chemo- and radio-therapy. CSCs are considered as the major cause of cancer relapse and recurrence after standard anti-proliferative therapy. Therefore, targeting cancer stem cells are expected to provide more effective therapeutic interventions to eradicate the cancer and prevent relapse (Batlle and Clevers, 2017; Reya et al., 2001; Wurth, 2016). Interestingly, CSCs also express CXCR4, and CXCR4 directs the trafficking and metastasis of these cells to the CXCL12-rich microenvironments such as bone marrow and subventricular zone in the brain that favor cancer stem cell maintenance, survival and growth. CXCR4 antagonists have been shown to mobilize CSCs from these protective microenvironments, and sensitize them to conventional chemo- and radiotherapy, and anti-angiogenic therapy (Burger and Kipps, 2006; Burger and Peled, 2009; Furusato et al., 2010; Redondo-Munoz et al., 2006; Walenkamp et al., 2017; Wurth, 2016).
Increasing evidence showed that tumor mass contains various cell types such as stromal fibroblasts, immune cells, endothelial cells, connective tissue, and extracellular matrix in addition to cancer cells that constitute tumor microenvironment (TME) or cancer cell niches. CXCR4/CXCL12 axis plays pivotal roles in tumor cell-microenvironment interaction that support tumor structure, growth, angiogenesis, and evasion of immune surveillance in various cancers of both the hematopoietic and nonhematopoietic system (Burger and Kipps, 2006; Burger and Peled, 2009; Walenkamp et al., 2017). CXCL12 can promote tumor angiogenesis by recruiting endothelial cells to the TME directly, or indirectly by attracting CXCR4-positive inflammatory cells to the tumor mass, and making them to secret proangiogenic factors (Owen and Mohamadzadeh, 2013; Walenkamp et al., 2017).
Growing evidence indicates that CXCR4/CXCL12 axis contributes to the lack of tumor responsiveness to angiogenesis inhibitors. Vascular endothelial growth factor (VEGF) was considered as the major pro-angiogenic factor in cancer, and was targeted for anti-angiogenic therapy in patients with rectal carcinoma using an anti-VEGF antibody bevacizumab (Genentech). Surprisingly, bevacizumab increased the expression of CXCL12 and CXCR4 in cancer cells, and increased plasma levels of CXCL12 in these patients were associated with rapid disease progression and metastasis (Owen and Mohamadzadeh, 2013; Xu et al., 2009). Therefore, the efficacy of a combination therapy using bevacizumab and plerixafor was evaluated for recurrent glioma (ClinicalTrials.gov identifier: NCT01339039). However, the study was terminated due to a low accrual rate (Walenkamp et al., 2017).
Inhibition of CXCR4/CXCL12 axis has been demonstrated to disrupt the tumor microenvironment (TME) and expose the tumor cells to immune attack by decreasing the infiltration of myeloid-derived suppressor cells, by increasing the ratio of CD8+ cytotoxic T cells to Treg cells, or eliminating tumor re-vascularization (Burger et al., 2011; Domanska et al., 2013; Walenkamp et al., 2017). Administration of CXCR4 antagonists, AMD3100 or T22, acted synergistically with immune checkpoint inhibitors such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) antibodies, programmed cell death protein 1 (PD-1), and programmed cell death ligand 1 (PD-L1) antibodies and greatly increased their antitumor activity in a model of pancreatic ductal adenocarcinoma, advanced hepatocellular carcinoma (HCC), and CXCR4-transduced B16 melanoma (Chen et al., 2015; Feig et al., 2013; Lee et al., 2006; Scala, 2015; Walenkamp et al., 2017). These results indicate that targeting CXCR4/CXCL12 axis offers benefit to the conventional immune checkpoint inhibitors.
Various drugs targeting CXCR4 have been developed (Peled, et al., 2012; Debnath, et al., 2013; Walenkamp, et al., 2017). CXCR4 inhibitors can be divided into 5 categories:                (1) non-peptide small molecule antagonists, such as AMD3100 (plerixafor, Mozobil™, Genzyme (MA, USA); (Cashen et al., 2007; Donzella et al., 1998), AMD070 (AMD11070; Crawford and Alan Kaller, 2008; Stone et al., 2007), AMD3465 (Genzyme Corp., Biochem Pharmacol. 2009 Oct. 15; 78(8):993-1000; Bodart et al., 2009; Ling et al., 2013), GSK812397 (Jenkinson et al., 2010; Planesas et al., 2015), KRH-3955 (Murakami et al., 2009; Nakasone et al., 2013), KRH-1636 (Ichiyama et al., 2003), D-[Lys3] GHRP-6 (Patel et al., 2012), TG-0054 (Burixafor, TaiGen Biotechnology Co., Ltd.; de Nigris et al., 2012; Hsu et al., 2015), WZ811 (Zhan et al., 2007), MSX-122 (Metastatix, Inc.; Liang et al., 2012), and 508MCl (Compound 26; Zhu et al., 2010);        (2) small-peptide antagonists, such as anti-HIV peptides T22 (Masuda et al., 1992), T134 and T140 (Tamamura et al., 1998), BKT140 (a/k/a BL-8040; TF14016; 4F-Benzoyl-TN14003, Biokine Therapeutics Ltd. (Rehovot, Israel); Fahham et al., 2012; Otani et al., 2012), ALX40-4C (Canadian company Allelix Biopharmaceuticals (ON, Canada); Doranz et al., 2001), GST-NT21MP (Yang et al., 2014), FC131 (Tanaka et al., 2009; Tanaka et al., 2008), FC122 (Inokuchi et al., 2011), POL6326 (de Nigris et al., 2012), and LY2510924 (Lilly) (Peng et al., 2015);        (3) antibodies to CXCR4, such as ulocuplumab (MDX1338/BMS-936564; Kuhne et al., 2013), PF-06747143 (Pfizer; Liu et al., 2017), 12G5 (Endres et al., 1996), i-bodies (AD-114, AD-114-6H, AD-114-Im7-FH, and AD-114-PA600-6H; AdAlta; Griffiths et al., 2016; Griffiths et al., 2018), nanobodies (238D2 and 238D4; Jahnichen et al., 2010; Proc. Natl Acad. Sci. 2010, USA 107(47), 20565-20570), and ALX-0651 (Ablynx, biparatopic nanobody, ClinicalTrials.gov Identifier: NCT01374503);        (4) ligand (CXCL12) analogs with suppressive activity, such as CTCE-9908 (Chemokine Therapeutics (BC, Canada); Wong et al., 2014); and        (5) radiolabeled CXCR4 ligands, such as [99mTc]O2-AMD3100 (Hartimath et al., 2013), [68Ga]pentixafor (Demmer et al., 2011; Gourni et al., 2011), [177Lu]pentixather, and [90Y]pentixather (Herrmann et al., 2016).        
AMD3100 (JM 3100, plerixafor, marketed as Mozobil) is a CXCR4-specific small molecule antagonist that inhibits CXCL12-mediated calcium mobilization and chemotaxis in various cell types (Hatse, Princen et al. 2002), and prevents tumor growth in mouse xenograft models (Rubin, Kung et al. 2003, Cho, Yoon et al. 2013, Liao, Fu et al. 2015). AMD3100 was originally developed for HIV therapy (as an HIV entry blocker that specifically antagonizes CXCR4, one of the HIV entry co-receptors), but was approved by the U.S. Food and Drug Administration only for stem cell mobilization in patients with lymphoma and multiple myeloma in 2008 (Keating 2011). During the initial clinical trial of AMD3100 for HIV infection, this compound was noted to cause hematopoietic stem cell (HSC, CD34+) mobilization from bone marrow to peripheral blood (Broxmeyer et al., 2005; De Clercq, 2003; Liles et al., 2003).
Development of AMD3100 for HIV therapy was suspended due to significant side effects including thrombocytopenia, premature ventricular contractions, and leukocytosis following long-term inhibition of CXCR4/CXCL12 axis (Hendrix, et al., 2004; Peled, et al., 2012). Although investigating the possibility of the use of AMD3100 as an anticancer drug is underway, lack of oral availability and some severe side effects accompanying with long-term use need to be overcome (Peled, et al., 2012). Instead, subcutaneous AMD3100 was approved in combination with G-CSF for autologous stem cell mobilization and transplantation for patients in the US with non-Hodgkin lymphoma (NHL) or multiple mylenoma for up to 4 consecutive days, and for patients in Europe with multiple mylenoma or lymphoma for 2-4 consecutive days and for up to 7 consecutive days, respectively (DiPersio et al., 2009a; DiPersio et al., 2009b; Keating, 2011). So far, AMD3100 is the only CXCR4 antagonist approved by the FDA.
AMD3100 has been shown to inhibit CXCL12-mediated calcium mobilization and chemotaxis in various cancer cell types expressing CXCR4 (Hatse et al., 2002), and has demonstrated significant anti-tumor activity with decreased metastasis and increased overall survival in various mouse xenograft models (Burger et al., 2011; Chatterjee et al., 2014; Cho et al., 2013; Debnath et al., 2013; Domanska et al., 2013; Liao et al., 2015; Rubin et al., 2003; Walenkamp et al., 2017).
However, AMD3100 exhibits partial agonism for CXCR4 in vitro and increases the proliferation of meloma cells (Kim et al., 2010; Zhang et al., 2002). AMD3100 also acts as a positive allosteric modulator of CXCR7, an alternative chemokine receptor for CXCL12, and increases CXCL12 binding to CXCR7 (Kalatskaya et al., 2009). Like CXCR4, CXCR7 is also highly expressed by many types of cancers and is associated with tumor metastasis (Decaillot et al., 2011; Zabel et al., 2011). Due to the complex properties of AMD3100, the exact role of AMD3100 on cancer needs to be investigated carefully.
AMD3100 has been clinically evaluated as anticancer drug in combination with conventional cytotoxic drugs such as mitoxantrone, etoposide, cytarabine, daunorubicin, azacitidine, lenalidomide, decitabine, clofarabine, fludarabine, and/or idarubicin; receptor tyrosine kinase inhibitors AC220 and sorafenib; HSP90 inhibitor (Ganetespib); G-CSF; proteasome inhibitor (bortezomib); or monoclonal antibody (rituximab) in hematological malignancies including acute myeloid leukemia (AML), MM, Myelodysplastic Syndromes (MDS), CLL, and small lymphocytic lymphoma (SLL) (ClinicalTrials.gov Identifier: NCT00512252, NCT00694590, NCT00903968, NCT00990054, NCT01065129, NCT01373229, NCT01352650, NCT01236144, NCT01301963, NCT01160354, NCT01027923, NCT00943943, and NCT01435343). AMD3100 was examined for interrupting lymphoid tumor microenvironment communication (NCT01610999) and for chemosensitization in combination with other anti-cancer drugs in patients with relapsed or refractory AML, ALL, and MDS (ClinicalTrials.gov identifier: NCT00906945 and NCT01319864).
AMD3100 has been or is also being evaluated in solid tumors including high grade glioma, Ewing's sarcoma, neuroblastoma, pancreatic, ovarian, and colorectal cancers alone or in combination with angiogenesis inhibitor bevacizumab (ClinicalTrials.gov identifier: NCT01339039, NCT01977677, NCT01288573, NCT02179970, and NCT03277209).
AMD3100 has been tested or is currently being evaluated for the mobilization of the hematopoietic stem/progenitor/precursor cells in patients with various hematological malignancies and diseases including AML, MDS, neutropenia, beta-thalassemia, sickle cell disease, WHIMS, Fanconi anemia, Wiskott-Aldrich Syndrome, systemic mastocytosis ((Domanska et al., 2013), NCT01058993, NCT01206075, NCT03226691, NCT00967785, NCT02678533, NCT03019809, and NCT00001756). It has been or currently being evaluated for recruiting CD34+ cells, endothelial progenitor cells (EPC), and/or CD117+ progenitor cells in patients with diabetes, wounds, critical limb ischemia, chronic obstructive pulmonary disease (COPD), cystic fibrosis, pulmonary fibrosis (ClinicalTrials.gov identifier: NCT02056210, NCT02790957, NCT01916577, NCT03182426).
Although investigating the possibility of the use of AMD3100 as an anticancer drug is underway, lack of oral availability and some severe side effects accompanying with long-term use need to be overcome (Peled et al., 2012).
The orally available CXCR4 antagonist, AMD070 (also referred to herein as AMD-070, AMD11070, AMD-11070, or X4P-001; X4 Pharmaceuticals) has demonstrate antitumor activity in various tumor models (Morimoto et al., 2016; O'Boyle et al., 2013; Parameswaran et al., 2011) and has been studied in a phase I/II clinical trial in HIV infected subjects (ClinicalTrials.gov identifier: NCT00089466, (Debnath et al., 2013). AMD070 is now in Phase II/III clinical trial in patients with WHIM syndrome, advanced melanoma, and renal cell carcinoma alone or together with pembrolizumab, nivolumab, or axitinib (ClinicalTrials.gov identifier: NCT03005327, NCT02823405, NCT02923531, and NCT02667886).
The efficacy of small peptide antagonists of CXCR4, T140 and its analogs TN14003, TC14012, and BKT140 (also referred to herein as BL-8040, TF14016, 4F-Benzoyl-TN14003, BioLineRx, Ltd.) for blocking CXCR4 has been demonstrated in numerous preclinical studies including stem cell mobilization, small-cell lung cancer (SCLC), breast cancer, melanoma, AML, chronic myeloid leukemia, MM, pancreatic cancer, and rheumatoid arthritis (Burger et al., 2011). Currently, BKT140 is under clinical development for hematopoietic stem and progenitor cell mobilization in various hematopoietic malignancies (ClinicalTrials.gov identifier: NCT02502968, NCT03154827, NCT02763384, NCT02639559, and NCT02462252). In addition, BKT140 is in Phase II clinical trials for interfering tumor microenvironment interaction in subjects with AML in combination with cytarabine (ClinicalTrials.gov identifier: NCT02502968). It is also investigated in various cancers such as AML, gastric cancer, non-small-cell lung cancer (NSCLC), and metastatic pancreatic cancers together with immune checkpoint inhibitors such as Pembrolizumab (Keytruda, Merck) and Atezolizumab (Tecentriq, Genentech/Roche) (ClinicalTrials.gov Identifier: NCT03154827, NCT03281369, NCT03337698, NCT02907099, NCT03193190, and NCT02826486).
Similarly, CTCE-9908 (Chemokine Therapeutics Corp., Canada), a peptide inhibitor of CXCR4, reduced metastases in mouse models of osteosarcoma and melanoma (Kim et al., 2008). After phase I study in patients with advanced solid cancers, CTCE-9908 was designated as an orphan drug for the treatment of osteosarcoma by the FDA in 2005. There is no further development after phase I/II clinical trial has been completed in 2008 (Debnath, et al., 2013).
MSX-122 (Altiris Therapeutics), a small molecule CXCR4 antagonist, has been abandoned after clinical trial in patients with solid tumors (ClinicalTrials.gov identifier: NCT00591682). Two major setbacks have been reported in 2017 in the field of developing CXCR4 antagonists as anti-cancer drugs. Bristol-Myers Squibb (BMS) discontinued a phase I/II study of ulocuplumab (BMS-936564/MDX1338, (Kuhne et al., 2013), a fully human anti-CXCR4 antibody, in patients with solid tumors for lack of efficacy (https://seekingalpha.com/article/4057548-bristol-failure-makes-small-dent-cxcr4-blocking-approach?page=2). Also, Eli Lilly decided to abandon a few anti-cancer programs including LY2510924, which is a CXCR4 peptide antagonist (http://www.fiercebiotech.com/biotech/lilly-puts-two-thirds-mid-phase-cancer-pipeline-up-for-sale-major-shake-up-r-d-priorities) after a series of clinical trials in solid tumors (ClinicalTrials.gov identifier: NCT02737072, NCT01391130, and NCT01439568). These events indicate that development of CXCR4 antagonists as anti-cancer drugs, especially for solid tumors, is an ongoing challenge.
Other small molecule antagonists that are currently under clinical investigation, include:
USL311 (Upsher-Smith) is under phase I and phase II in patients with solid tumors and glioblastoma multiforme (GBM), respectively (ClinicalTrials.gov identifier: NCT02765165); GMI-1359 (Glycomimetics) is in phase I trial (ClinicalTrials.gov identifier: NCT02931214).
PF-06747143 (Pfizer), a humanized anti-CXCR4 antibody, is in phase I clinical trial in patients with AML either alone or combined with chemotherapy (ClinicalTrials.gov identifier: NCT02954653, (Liu et al., 2017)).
AD-114 (i-body, AdAlta; (Griffiths et al., 2018) is a humanized shark anti-CXCR4 antibody and received orphan drug designation from united states FDA for a drug candidate used for treatment of Idiopathic Pulmonary Fibrosis in 2017 (https://www.reuters.com/article/idUSFWN1F70Y0). AD-114 study is mainly focused on fibrotic conditions, including wet age-related macular degeneration and non-alcoholic fatty liver disease (https://lungdiseasenews.com/2017/08/23/adalta-present-research-on-investigative-therapy-ad-114-at-ipf-summit/).
POL6326 (balixafortide, Polyphor), a peptide CXCR4 antagonist, has been under clinical evaluation in subjects with breast cancer (ClinicalTrials.gov identifier: NCT01837095), hematologic malignancies (ClinicalTrials.gov identifier: NCT01413568), and stem cell mobilization and healing in patients with acute myocardial infarction (ClinicalTrials.gov identifier: NCT01905475).
Radiolabeled CXCR4 ligands such as [99mTc]O2-AMD3100 and [68Ga]Pentixafor has been used for preclinical or clinical SPECT or PET imaging of CXCR4 expression (Demmer et al., 2011; Gourni et al., 2011; Hartimath et al., 2013; Walenkamp et al., 2017). PET imaging with [68Ga]Pentixafor has demonstrated increased expression of CXCR4 not only in hematologic and solid tumors such as leukemia, lymphoma, MM, adrenocortical carcinoma, and SCLC, but also in other pathological conditions such as splenosis, stroke, atherosclerosis, and myocardial infarction (Walenkamp et al., 2017).
A peptide CXCR4 ligand labeled with α- or β-emitters ([177Lu]pentixather and [90Y]pentixather) has been tested in more than 30 therapies as CXCR4-directed endoradiotherapy together with standard chemotherapy in patients with hematologic malignancies (Walenkamp et al., 2017).
Although various drugs antagonizing CXCR4 or antibody targeting CXCR4 have been developed and are under investigation, be it alone, in combination with conventional anticancer therapies, or with an immune checkpoint inhibitor, success has been limited so far (Peled, et al., 2012, Debnath, et al., 2013, Walenkamp, et al., 2017). Due to the pivotal roles of CXCR4 in B and T lymphocyte development and immune surveillance, long-term or persistent inhibition of CXCR4 with CXCR4 antagonist would potentially evoke immune system and hematopoietic dysfunctions and expose cancer patients to risk of immune suppression (Burger, 2009). Hematopoietic stem cells (HSCs) are normally protected in bone marrow niches. If CXCR4 antagonists are administered with cytotoxic drugs or radiotherapy, HSCs mobilized into periphery would be exposed to the effects of cytotoxic treatment which could exacerbate cytopenias. Cardiac complications observed in patients treated with AMD3100 also raised general concern in prolonged use of CXCR4 antagonists as anticancer drugs.
To avoid potential side effects associated with conventional CXCR4 antagonists and to develop more efficient anti-cancer drugs targeting CXCR4, new paradigm for designing CXCR4 inhibitor is urgently required.
Recently, GPCR heteromers offer new possibilities for developing more specific and disease-restricted therapeutics. Traditionally, GPCRs were considered to be monomeric since monomeric GPCRs can activate G proteins by inducing conformational changes of the heptahelical domain upon ligand binding (Pin et al., 2008; Okada et al., 2001). However, increasing evidence demonstrates that GPCRs may form oligomers, either homo- or hetero-oligomers, and GPCR heteromers can exhibit specific properties that clearly distinguish them from existing well-defined monomers, such as signaling pathways, ligand binding affinities, internalization, and recycling (De Falco et al., 2007; Ferre et al., 2010; Gomes et al., 2016). Therefore, GPCR oligomerization may provide a way to increase the diversity of GPCR entities with a limited number of genes (Park and Palczewski, 2005). Thus, identification of novel GPCR heteromers would offer new possibilities for understanding the roles of GPCR heteromers in specific tissues and in specific diseases, as well as providing new opportunities for developing more efficient therapeutics with less side effects. (Milligan 2008; Rozenfeld and Devi 2010; Gomes et al., 2016; Farran 2017).
CXCR4 also forms heteromers with different GPCRs. So far CXCR4 is known to interact with GPCRs in the chemokine receptor family (CCR2 (Rodriguez-Frade et al., 2004; Sohy et al., 2007; Sohy et al., 2009; Armando et al., 2014), CCRS (Agrawal et al., 2004; Rodriguez-Frade et al., 2004; Sohy et al., 2007, Sohy et al., 2009; Martinez-Munoz et al., 2014), CXCR3 (Watts et al., 2013), and CXCR7 (Sierro et al., 2007; Levoye et al., 2009; Decaillot et al., 2011)), chemerin chemokine-like receptor 1 (CMKLR1) (de Poorter et al., 2013), δ-opioid receptor (OPRD) (Pello et al. 2008; Burbassi et al., 2010), and adrenergic receptor family (ADRA1A (Tripathi et al., 2015), ADRA1B (Tripathi et al., 2015), and ADRB2 (LaRocca et al. 2010; Nakai et al., 2014)).
The existence of CXCR4-CCR2 and CXCR4-CCR5 heteromers was first noticed by studying HIV infection. Rodriguez-Frade et al. showed that CCR2-01, a CCR2-specific monoclonal antibody, did not compete with CCL2 for binding to CCR2 or trigger CCR2 signaling, but blocked replication of monotropic (R5) and T-tropic (X4) HIV strains by inducing oligomerization of CCR2 with CCR5 or CXCR4 (Rodriguez-Frade et al., 2004). Agrawal et al. also showed that CCR5Δ32 specifically inhibited CCR5 and CXCR4 cell surface expression by heteromerization with CCR5 and CXCR4, thereby inhibiting HIV coreceptor activity of CCR5 and CXCR4 in CD4+ cells (Agrawal et al., 2004). Co-expression of CCR5 was shown to prevent HIV-1 gp120 binding to the cell surface and to reduce X4 HIV-1 infectivity by inducing CCR5-CD4-CXCR4 oligomerization and conformational changes in CD4 and CXCR4 (Martinez-Munoz et al., 2014).
Sohy et al. reported the existence of negative binding cooperativity between the subunits of CXCR4-CCR2 and CXCR4-CCR5 heteromers, i.e. the ligands of one receptor competed for the binding of a specific tracer of the other, in recombinant cell lines and primary leukocytes (Sohy et al., 2007; Sohy et al., 2009). They also demonstrated that TAK-779, the CCR2 and CCR5 antagonist, prevented calcium mobilization and chemotaxis initiated by the CXCR4 agonist CXCL12 in cells co-expressing CCR2 and CXCR4 or CCR5 and CXCR4 (Sohy et al., 2007; Sohy et al., 2009). Armando et al. showed that CXCR4 and CCR2 can form homo- and hetero-oligomers, and co-activation of CCR2 and CXCR4 with the human monocyte chemotactic protein 1 (MCP-1) and CXCL12 caused a synergistic increase in calcium mobilization (Armando et al., 2014).
Watts et al. identified CXCR4-CXCR3 heteromers in HEK293T cells and showed negative binding cooperativity for endogenous agonists and small CXCR3 agonist VUF10661, but not for CXCR3 antagonists VUF10085 nor CXCR4 antagonist AMD3100 (Watts et al., 2013).
CXCR4 also interacts with ACKR3, also known as CXCR7, a chemokine family GPCR that binds the CXCL12 and CXCL11, but is unable to couple with G proteins upon agonist binding. Sierro et al. identified CXCR4-ACKR3 heteromers as functional heteromers that displayed enhanced CXCL12-induced calcium signaling and altered ERK1/2 signaling in HEK293 cells (Sierro et al., 2007). Lovoye et al., reported an opposite result showing that CXCR4-ACKR3 heteromers exhibited reduced Gai and calcium responses upon exposure to CXCL12 in CHO-K1 cells (Levoye et al., 2009). Decaillot et al. noticed that co-expression of CXCR4 and ACKR3 constitutively recruited β-arrestin to CXCR4/ACKR3 heteromers and potentiated CXCL12-mediated β-arrestin-dependent downstream signaling pathways, including ERK1/2, p38 MAPK, and enhanced cell migration upon exposure to CXCL12 (Decaillot et al., 2011).
CXCR4 also interacts with the chemerin chemokine-like receptor 1 (CMKLR1, also known as ChemR23) (de Poorter et al., 2013). Although CXCR4-CMKLR1 heteromers display negative agonist binding cooperativity similar to CXCR4-CCR2 and CXCR4-CCR5 heteromers reported by Sohy et al. (Sohy et al., 2007; Sohy et al., 2009), AMD3100 did not cross inhibit chemerin binding or inhibit calcium mobilization induced by CXCL12 (de Poorter et al., 2013).
CXCR4 and the δ-opioid receptor (DOR) are widely distributed in brain tissues and immune cells. Pello et al. reported that CXCR4 and DOR can form heteromers, and simultaneous stimulation with both agonists suppressed the activation of Gai signaling, and prevented cell migration toward CXCL12, although individual agonist elicit robust Gai signaling (Pello et al., 2008). Burbassi et al. also noticed increased CXCR4-DOR heteromers and reduced coupling of CXCR4 to G-proteins in brain tissue and cultured glia from μ-opioid receptor (MOR)-deficient mice (Burbassi et al., 2010). CXCR4 function was rescued with a DOR antagonist, indicating that DOR is involved in the suppression of CXCR4 in glia by forming CXCR4-DOR heteromers (Burbassi et al., 2010).
CXCR4 is also known to interact with α- and β-adrenergic receptors (α-AR and β-AR). LaRocca et al. reported that stimulation of CXCR4 with CXCL12 negatively regulates β-AR-induced cAMP accumulation and PKA-dependent phosphorylation of phospholamban in adult rat ventricular myocytes using non-selective β-AR agonist, isoproterenol (LaRocca et al., 2010). They showed the co-expression of CXCR4 and ADRB2 (β2-AR) on the cardiac myocytes, and physical association of CXCR4 with ADRB2 using co-immunoprecipitation and bioluminescence resonance energy transfer, suggesting the CXCR4-ADRB2 heteromer as a novel cardiac modulator.
Nakai et al. studied the function of ADRB2 on lymphocytes. Stimulation of lymphocytes with ADRB2-selective agonists suppressed egress of lymphocytes from lymph nodes and produced lymphopenia in mice (Nakai et al., 2014). ADRB2 physically interacted with CCR7 and CXCR4, and activation of ADRB2 enhanced retention-promoting signals through CCR7-ADRB2 and CXCR4-ADRB2 heteromers, and subsequently reduced lymphocyte egress from lymph nodes.
Tripathi et al. revealed the presence of CXCR4-ADRA1A (α1A-AR) and CXCR4-ADRA1B (α1B-AR) heteromers on the surface of vascular smooth muscle cells (VSMC) (Tripathi et al., 2015). A peptide derived from the second transmembrane helix of CXCR4 disrupted the interaction between ADRA1A/B and CXCR4, inhibited calcium mobilization and contraction of VSMC upon α1-AR stimulation. Activation of CXCR4 with CXCL12 increased the potency of α1-AR agonists on blood pressure response in rats, suggesting that CXCR4-ADRA1A/B heteromers could be a novel pharmacological target for blood pressure regulation.
CXCR4 was reported to interact with CNR2 (Cannabinoid Receptor 2, aka CB2) (Coke et al., 2016; Scarlett et al., 2018). Simultaneous activation of CXCR4 and CB2 with both agonists resulted in reduction of ERK1/2 activation, calcium mobilization and cellular chemotaxis. These results show that cannabinoid system can negatively modulate CXCR4 function and tumor progression.
As described above, GPCRs forming heteromers with CXCR4 have been studied within a limited number of GPCR families, such as chemokine, adrenergic, and opioid receptor families. Considering the major role and increased expression of CXCR4 in a variety of pathological conditions, there is a great potential for the existence of different CXCR4-GPCRx heteromers that confer unique features to a specific disease. However, due to the large number of GPCR genes (about 800) and difficulties in establishing high-throughput proximity-based screening techniques and functional assays, the identification of new CXCR4-GPCRx heteromers has been a major challenge.
Thus, there exists in the art a need for identifying functional GPCR heteromers, such as CXCR4-GPCRx, and developing their inhibitors for use as GPCR heteromer-targeting cancer therapeutics with higher efficacy and lower side effects. This invention satisfies this need and provides related advantages.